Fluorescence-based high throughput screening assays for protein kinases and phosphatases

ABSTRACT

The invention relates to novel fluorescence-based assays for protein kinases and phosphatases which can be used in high throughput screening. The methods of the invention utilize a competitive immunoassay to determine the amount of substrate that is phosphorylated or dephosphorylated during the course of a kinase or phosphatase reaction to yield a product, as well as the phosphorylating or dephosphorylating activity of a kinase or phosphatase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Application Ser. No. 09/731,428filed Dec. 6, 2000 now U.S. Pat. No. 6,630,311, which is a divisional ofU.S. application Ser. No. 09/204,335 now U.S. Pat. No. 6,203,994 filedDec. 2, 1998, which claims the benefit of U.S. Provisional ApplicationNo. 60/067,833, filed 5 Dec. 1997.

FIELD OF THE INVENTION

The invention relates to novel fluorescence-based assays for kinases andphosphatases which can be used in high throughput screening.

BACKGROUND OF THE INVENTION

Eukaryotes employ phosphorylation and dephosphorylation of specificproteins to regulate many cellular processes (T. Hunter, Cell 80:225-236(1995); (Karin, M., Curr. Opin. Cell Biol. 3:467-473 (1991)). Theseprocesses include signal transduction, cell division, and initiation ofgene transcription. Thus, significant events in an organism'smaintenance, adaptation, and susceptibility to disease are controlled byprotein phosphorylation and dephosphorylation. These phenomena are soextensive that it has been estimated that humans have around 2,000protein kinase genes and 1,000 protein phosphatase genes (T. Hunter,Cell 80:225-236 (1995)), some of these likely coding for diseasesusceptibility. For these reasons, protein kinases and phosphatases aregood targets for the development of drug therapies.

The most frequently used protein kinase and phosphatase screens employeither radioactive ATP or ELISAs. However, the use of radioactive ATP isundesirable due to the attendant costs of record-keeping,waste-disposal, and the fact that the assay format is not homogeneous.ELISAs are undesirable because they have a lower assay throughput due tothe extra steps required for both washing and the enzyme reaction.

Fluorescence detection in the visible wavelengths offer an alternativeto the use of radiotracers or ELISAs for kinase and phosphatase assays,as fluorescence offers detection limits comparable to those ofradioactivity. Furthermore, this eliminates the cost of radioactivewaste disposal. For example, the change in absorbance and fluorescencespectra of phosphotyrosine which occurs upon dephosphorylation has beenused for the continuous monitoring of protein-tyrosine phosphatase (PTP)activity (Zhao, Z. et al., Anal. Biochem. 202:361-366 (1993)). However,previously developed fluorometric assays for kinases and phosphataseshave not been especially amenable to the requirements of high throughputscreening.

Fluorescence detection frequently offers the advantage of usinghomogeneous assay formats (i.e.—“mix, incubate, and read”). Indeed, thehigh throughput screening (HTS) field is moving rapidly toward the useof fluorescence, luminescence, absorbance, and other optical methods.Two fluorescence techniques, fluorescence polarization (FP) andfluorescence resonance energy transfer (FRET) are finding widespread usefor assays, both in the private sector for HTS, secondary assaysincluding kinetics, SAR studies, etc., and in university laboratories.The use of FP is particularly desirable since its readout is independentof the emission intensity (Checovich, W. J., et al., Nature 375:254-256(1995); Dandliker, W. B., et al., Methods in Enzymology 74:3-28 (1981))and is thus insensitive to the presence of colored compounds that quenchfluorescence emission. FRET, although susceptible to quenching, can alsobe used effectively, especially for continuous enzyme assays.

From the forgoing, it will be clear that there is a continuing need forthe development of cost-effective, facile, and sensitive optical kinaseand phosphatase assays for both high throughput screening (HTS) andsecondary assays.

Information Disclosure

Checovich, W. J., et al., Nature 375:254-256 (1995).

Dandliker, W. B., et al., Methods in Enzymology 74:3-28 (1981).

E. Harlow and D. Lane, eds., Antibodies A Laboratory Manual, Cold SpringHarbor Laboratory (1988).

T. Hunter, Cell 80:225-236 (1995).

Leavine, L. M., et al., Anal. Biochem. 247:83-88 (1997).

Owicki, J. C., Genetic Engineering News 17:27 (Nov. 1, 1997).

Rotman, B., et al., Proc. Nat. Acad. Sci. 50:1-6 (1963).

Seethala, R. and R. Menzel, A Fluorescence Polarization Tyrosine KinaseAssay for High Throughput Screening, 3rd Annual Conference of TheSociety for Biomolecular Screening, San Diego, Calif., Sep. 22-25, 1997.

SUMMARY OF THE INVENTION

The invention relates to novel fluorescence-based assays for proteinkinases and phosphatases which can be used in high throughput screening.The methods of the invention utilize a competitive immunoassay todetermine the amount of substrate that is phosphorylated ordephosphorylated during the course of a kinase or phosphatase reactionto yield a product, as well as the phosphorylating or dephosphorylatingactivity of a kinase or phosphatase.

Thus, in one embodiment, the invention relates to a method ofdetermining the phosphorylating activity of an enzyme comprising thesteps of:

(a) combining the enzyme with

(i) a reporter molecule comprising a fluorescent label and aphosphorylated amino acid, wherein the amino acid is selected from thegroup consisting of serine, threonine and tyrosine;

(ii) a substrate molecule comprising the same amino acid that isphosphorylated in said reporter, wherein said substrate molecule iscapable of being phosphorylated at said amino acid by said enzyme toyield a product;

(iii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid; and

(iv) a high-energy phosphate source;

(b) measuring the fluorescence polarization (FP), FQ, or fluorescenceresonance spectroscopy (FCS) of the reporter following the combinationof step (a); and

(c) using the FP, FQ, or FCS measurement of step (b) to determine theactivity of the enzyme.

In another embodiment, the invention relates to a method for determiningthe dephosphorylating activity of an enzyme comprising the steps of:

(a) combining the enzyme with

(i) a reporter molecule comprising a fluorescent label and aphosphorylated amino acid, wherein the amino acid is selected from thegroup consisting of serine, threonine and tyrosine;

(ii) a substrate molecule comprising the same phosphorylated amino acidas said reporter, wherein said substrate molecule is capable of beingdephosphorylated at said amino acid by said enzyme to yield a product;and

(iii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid;

(b) measuring the FP, FQ, or FCS of said reporter following thecombination of step (a); and

(c) using the FP, FQ, or FCS measurement of step (b) to determine theactivity of the enzyme.

The methods of the invention can also be used to determine thephosphorylation or dephosphorylation of a substrate molecule by anenzyme. Thus, in another embodiment, the invention relates to a methodfor determining the phosphorylation of a substrate molecule by an enzymeat an amino acid selected from the group consisting of serine, threonineand tyrosine, comprising the steps of:

(a) combining the substrate molecule with

(i) the enzyme

(ii) a reporter molecule comprising a fluorescent label and aphosphorylated amino acid, wherein the amino acid is the same amino acidwhich is phosphorylated in the reporter;

(iii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid; and

(iv) a high-energy phosphate source;

(b) measuring the FP, FQ, or FCS of the reporter following thecombination of step (a); and

(c) using the FP, FQ, or FCS measurement of step (b) to determinewhether the substrate molecule has been phosphorylated.

In another embodiment, the invention relates to a method for determiningthe dephosphorylation of a substrate molecule by an enzyme, wherein thesubstrate molecule comprises a phosphorylated amino acid, and whereinthe amino acid is selected from the group consisting of serine,threonine and tyrosine, comprising the steps of:

(a) combining the substrate molecule with

(i) the enzyme;

(ii) a reporter molecule comprising a fluorescent label and aphosphorylated amino acid, wherein the reporter molecule comprises thesame phosphorylated amino acid as the substrate molecule; and

(iii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid;

(b) measuring the FP, FQ, or FCS of the reporter following thecombination of step (a); and

(c) using the FP, FQ, or FCS measurement of step (b) to determinewhether the substrate molecule has been dephosphorylated.

In a preferred embodiment, the substrate in any of the above methods iscombined with the enzyme before the addition of the reporter and theantibody. In another preferred embodiment, the substrate, the reporter,and the antibody are combined with the enzyme simultaneously.

Because the above-described methods of the invention utilize acompetitive immunoassay to determine the amount of phosphorylated ordephosphorylated substrate (i.e., the amount of product) produced, theamount of phosphorylated substrate required to displace the reporterfrom the antibody will vary depending upon the K_(d) of thephosphorylated substrate for the antibody and the K_(d) of the antibodyfor the reporter molecule.

Thus, where the K_(d) of the phosphorylated substrate for the antibodyis, e.g., 10-fold higher than the K_(d) of the antibody for the reportermolecule, then an amount of phosphorylated substrate ten times higherthan the amount of reporter will be required for the phosphorylatedsubstrate to displace the reporter from the antibody.

In a more preferred embodiment, the K_(d) of the phosphorylatedsubstrate for the antibody will be approximately equal to the K_(d) ofthe antibody for the reporter molecule. In a still more preferredembodiment, the K_(d) of the phosphorylated substrate for the antibodywill be less than the K_(d) of the antibody for the reporter molecule.In this situation, phosphorylation of the substrate will quantitativelydisplace the reporter from the antibody.

In accordance with the above description, one way of reducing the amountof substrate needed to displace the reporter from the antibody is tochoose a reporter having a low K_(d) for the antibody. Becauseanti-phosphorylamino acid antibodies may have a higher affinity for afluorescently labeled phosphorylamino acid than for a fluorescentlylabeled peptide comprising the same phosphorylamino acid, in a preferredembodiment, such peptides are used as the reporter.

The methods of the invention also allow the utilization of a continuousrecording assy (i.e., a “real time” assay) for the determination ofeither kinase or phosphatase activity by using a FRET format.

Thus, in another embodiment, the invention relates to a method ofdetermining the phosphorylating activity of an enzyme comprising thesteps of:

(a) combining the enzyme with:

(i) a substrate molecule comprising an amino acid selected from thegroup consisting of Ser, Thr, and Tyr, wherein said substrate moleculeis capable of being phosphorylated at said amino acid by said enzyme toyield a product, and wherein said substrate molecule is labeled with anacceptor fluorophore;

(ii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid, said antibody being labeled with a donorfluorophore which corresponds to the acceptor fluorophore labeling saidsubstrate; and

(iii) a high-energy phosphate source;

(b) measuring the FRET of the combination of step (a); and

(c) using the FRET measurement of step (b) to determine the activity ofthe enzyme.

In another embodiment, the invention relates to a method of determiningthe phosphorylating activity of an enzyme comprising the steps of:

(a) combining the enzyme with:

(i) a substrate molecule comprising an amino acid selected from thegroup consisting of Ser, Thr, and Tyr, wherein said substrate moleculeis capable of being phosphorylated at said amino acid by said enzyme toyield a product, and wherein said substrate molecule is labeled with adonor fluorophore;

(ii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid, said antibody being labeled with an acceptorfluorophore which corresponds to the donor fluorophore labeling saidsubstrate; and

(iii) a high-energy phosphate source;

(b) measuring the FRET of the combination of step (a); and

(c) using the FRET measurement of step (b) to determine the activity ofthe enzyme.

In another embodiment, the invention relates to a method of determiningthe dephosphorylating activity of an enzyme comprising the steps of:

(a) combining the enzyme with:

(i) a substrate molecule comprising a phosphorylated amino acid selectedfrom the group consisting of phosphoserine, phospothreonine andphosphotyrosine, wherein said substrate molecule is labeled with anacceptor fluorophore;

(ii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid, said antibody being labeled with a donorfluorophore which corresponds to the acceptor fluorophore labeling saidsubstrate; and

(iii) a high-energy phosphate source;

(b) measuring the FRET of the combination of step (a); and

(c) using the FRET measurement of step (b) to determine the activity ofthe enzyme.

In another embodiment, the invention relates to a method of determiningthe dephosphorylating activity of an enzyme comprising the steps of:

(a) combining the enzyme with:

(i) a substrate molecule comprising a phosphorylated amino acid selectedfrom the group consisting of phosphoserine, phospohthreonine andphosphotyrosine, wherein said substrate molecule is labeled with a donorfluorophore;

(ii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid, said antibody being labeled with an acceptorfluorophore which corresponds to the donor fluorophore labeling saidsubstrate; and

(iii) a high-energy phosphate source;

(b) measuring the FRET of the combination of step (a); and

(c) using the FRET measurement of step (b) to determine the activity ofthe enzyme.

In another embodiment, the invention relates to a method for determiningthe phosphorylation of a substrate molecule by an enzyme at an aminoacid selected from the group consisting of serine, threonine andtyrosine, wherein said substrate molecule is labeled with an acceptorfluorophore, comprising the steps of:

(a) combining the substrate molecule with

(i) the enzyme

(ii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid, said antibody being labeled with a donorfluorophore which corresponds to the acceptor fluorophore labeling saidsubstrate; and

(iii) a high-energy phosphate source;

(b) measuring the FRET of the reporter following the combination of step(a); and

(c) using the FRET measurement of step (b) to determine whether thesubstrate molecule has been phosphorylated.

In another embodiment, the invention relates to a method for determiningthe phosphorylation of a substrate molecule by an enzyme at an aminoacid selected from the group consisting of serine, threonine andtyrosine, wherein said substrate molecule is labeled with a donorfluorophore, comprising the steps of:

(a) combining the substrate molecule with:

(i) the enzyme

(ii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid, said antibody being labeled with an acceptorfluorophore which corresponds to the donor fluorophore labeling saidsubstrate; and

(iii) a high-energy phosphate source;

(b) measuring the FRET of the reporter following the combination of step(a); and

(c) using the FRET measurement of step (b) to determine whether thesubstrate molecule has been phosphorylated.

In another embodiment, the invention relates to a method for determiningthe dephosphorylation of a substrate molecule by an enzyme, wherein thesubstrate molecule comprises a phosphorylated amino acid selected fromthe group consisting of phosphoserine, phospohthreonine andphosphotyrosine, and wherein said substrate molecule is labeled with anacceptor fluorophore comprising the steps of:

(a) combining the substrate molecule with:

(i) the enzyme;

(ii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid, said antibody being labeled with a donorfluorophore which corresponds to the acceptor fluorophore labeling saidsubstrate;

(b) measuring the FRET of the reporter following the combination of step(a); and

(c) using the FRET measurement of step (b) to determine whether thesubstrate molecule has been dephosphorylated.

In another embodiment, the invention relates to a method for determiningthe dephosphorylation of a substrate molecule by an enzyme, wherein thesubstrate molecule comprises a phosphorylated amino acid selected fromthe group consisting of serine, threonine and tyrosine, and wherein saidsubstrate molecule is labeled with a donor fluorophore comprising thesteps of:

(a) combining the substrate molecule with:

(i) the enzyme;

(ii) an antibody which selectively binds to a molecule comprising thephosphorylated amino acid, said antibody being labeled with an acceptorfluorophore which corresponds to the donor fluorophore labeling saidsubstrate;

(b) measuring the FRET of the reporter following the combination of step(a); and

(c) using the FRET measurement of step (b) to determine whether thesubstrate molecule has been dephosphorylated.

The methods of the invention can also be used to identify an agentcapable of increasing or decreasing the phosphorylating activity of anenzyme comprising the steps of:

(a) performing the above method of determining the phosphorylatingactivity of an enzyme in the presence and in the absence of the agent;

(b) comparing the activity of the enzyme in the presence of the agentwith the activity of the enzyme in the absence of the agent to determinewhether the phosphorylating activity of the enzyme in the presence ofthe agent is increased or decreased.

In yet another embodiment, the invention relates to a method ofscreening for an agent capable of increasing or decreasing thedephosphorylating activity of an enzyme comprising the steps of:

(a) performing the above method of determining the dephosphorylatingactivity of an enzyme in the presence and in the absence of said agent;

(b) comparing the activity of said enzyme in the presence of said agentwith the activity of said enzyme in the absence of said agent todetermine whether the dephosphorylating activity of said enzyme in thepresence of said agent is increased or decreased.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structures of reagents used in Example 1.

FIGS. 2A, 2B and 2C: FIGS. 2A, 2B, and 2C are graphs showing the bindingof sigma antibodies to fluoresceinated phosphorylamino acids. Fixedconcentrations of P-Tyr-F (25.8 nM)(FIG. 2A), P-Ser-F (5.9 nM)(FIG. 2B),or P-Thr-F (10 nM)(FIG. 2C) were titrated with the specific antibodiesas described in Methods of Example 1. The polarization was recordedafter each addition and the data analyzed using Equation 7 of Example 1in conjunction with a nonlinear least squares fitting program. In thisand subsequent figures, the solid lines represent the theoretical fitsto the experimental data.

FIGS. 3A and 3B: FIGS. 3A and 3B are graphs showing the binding of MBLanti-phosphotyrosine antibody to P-Tyr-F. A fixed concentration (15 nM)of P-Tyr-F was titrated with the MBL antibody as described in Methods ofExample 1. The FP (FIG. 3A) and total polarized emission (FIG. 3B) weremonitored during the titration. The quenching data were analyzed bynonlinear least squares fitting using Equation 7 of Example 1, and theFP data using Equation 20 of Example 1 with the values of C andQ_(c)/Q_(L) calculated from Equation 7 of Example 1 substituted into it.

FIGS. 4A, 4B and 4C: FIGS. 4A, 4B and 4C are graphs showing thedisplacement of fluoresceinated phosphorylamino acids from Sigmaantibodies by the corresponding unlabelled phosphorylamino acids.Concentrations of the fluoresceinated phosphorylamino acids and thecorresponding Sigma antibodies were fixed, and their displacement by thephosphorylamino acids was followed by measuring the decrease in thefluorescence polarization. The fixed concentrations were: FIG. 4A:P-Tyr-F, 1 nM and MAB, 2.05 nM sites; FIG. 4B: P-Ser-F, 1 nM and MAB, 1nM sites; FIG. 4C: P-Thr-F, 150 nM and MAb, 150 nM sites.

FIGS. 5A and 5B: FIGS. 5A and 5B are graphs showing the competitivedisplacement of P-Tyr-F from MBL antibody by phosphoryltyrosine asmeasured by FQ (FIG. 5A) and FP (FIG. 5B). MBL antibody, 7.1 nM sitesand P-Tyr-F, 1.7 nM were preincubated, and displacement initiated by theaddition of a concentrated solution of phosphotyrosine. The increase inemission intensity and decrease in FP were monitored as a function ofadded phosphoryltyrosine. The data were analyzed as described in FIG. 3.

FIG. 6 is a graph showing the competitive displacement of P-Tyr-F fromSigma antibody by phosphorylated JAK-2 kinase peptides. Sigma antibody,2.05 nM sites and P-Tyr-F, 1 nM, were preincubated and displacementinitiated as described for FIG. 5. The polarization data were analyzedas described in FIG. 3. --JAK-2(a); ∘--∘, JAK-2(b).

FIGS. 7A, 7B, 7C, and 7D: FIGS. 7A, 7B, 7C, and 7D are graphs showingthe competitive displacement of P-Tyr-F from MBL antibody byphosphorylated JAK-2 peptide substrates JAK-2(a) (FIGS. 7A and 7B) andJAK-2(b) (FIGS. 7C and 7D). All procedures were as described in FIG. 5.For both peptides, fixed antibody=7.1 nM sites and P-Tyr-F=1.7 nM.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to novel fluorescence-based assays for kinases andphosphatases which can be used in high throughput screening. As usedherein, the term “kinase” refers to an enzyme capable of phosphorylatingits substrate at a Ser, Thr, or Tyr residue, while the term“phosphatase” refers to an enzyme capable of dephosphorylating itssubstrate at a phosphoserine, phosphothreonine, or phosphotyrosineresidue. The methods of the invention utilize a competitive immunoassayto determine the amount of phosphorylated or dephosphorylated substrateproduced during the course of a kinase or phosphatase reaction, as wellas the phosphorylating or dephosphorylating activity of a kinase orphosphatase. Unless otherwise indicated, “phosphorylating activity” asused herein is synonymous with “kinase activity,” and “dephosphorylatingactivity” as used herein is synonymous with “phosphatase activity.”Similarly, unless otherwise indicated, a “kinase” is defined herein as abiological material capable of phosphorylating a peptide or protein, anda “phosphatase” is defined herein as a biological material capable ofdephosphorylating a peptide or protein. Further, where the enzyme usedin any of the assays of the invention is a kinase, the term“phosphorylated substrate” is synonymous with “product,” (i.e, theproduct derived from the enzymatic reaction). Similarly, where theenzyme used in any of the assays of the invention is a phosphatase, theterm “dephosphorylated substrate” is synonymous with “product,” (i.e,the product derived from the enzymatic reaction).

In the methods of the invention, a reporter molecule comprising afluorescent label and a phosphorylamino acid (P-AA) selected from thegroup consisting of Ser, Thr and Tyr (hereinafter referred to as a“reporter molecule,” or “reporter”) competes with a phosphorylatedsubstrate molecule, comprising the same P-AA as the reporter molecule,for an antibody specific for the P-AA. The antibody and reportermolecule are chosen so that binding of the antibody, to the reportercauses a change in the reporter which is detectable using FP, FQ, orFCS. Knowledge of the concentration of reporter and substrate used, thedissociation constant (K_(d)) of the phosphorylated substrate for theantibody, the K_(d) of the reporter for the antibody, and the change inthe fluorescent properties of the reporter will allow calculation of theamount of phosphorylated substrate present, and the determination ofkinase or phosphatase activity, as is described below.

Thus, by the methods of the invention, phosphorylation of a substratepeptide or protein by a kinase can be monitored by specific displacementof a reporter molecule from an antibody by the reaction product of thekinase assay (the phosphorylated substrate molecule). One assay formatfor the fluorescent kinase assay is given below.

In this new scheme, a kinase reaction is carried out in a reactionmixture by contacting a kinase with a substrate molecule in the presenceof a high energy phosphate source such as ATP or GTP. The reaction isallowed to proceed for a period of time, and is stopped (for example, bythe addition of a metal chelator such as EDTA or EGTA). Subsequently,both antibody and reporter are added to the reaction mixture, wherebythe reaction product (the phosphorylated substrate molecule)specifically displaces the reporter molecule from the antibody.

In another embodiment, the antibody and reporter molecule are present attime=0, so that the phosphorylated substrate competes with the reporterfor the antibody, giving intermediate values of polarization and/orquenching, thus providing a homogeneous format for high-throughputscreening. Of course, one of ordinary skill will realize that theantibody and reporter molecule can only be present at the time that theenzyme is added where neither the antibody nor the reporter are asubstrate for the enzyme.

Thus, the invention provides a method of determining the phosphorylatingactivity of an enzyme comprising the steps of:

(a) combining said enzyme with

(i) a reporter molecule comprising a fluorescent label and aphosphorylated amino acid, wherein said amino acid is selected from thegroup consisting of serine, threonine and tyrosine;

(ii) a substrate capable of being phosphorylated by said enzyme at thesame amino acid which is phosphorylated in said reporter;

(iii) an anti-phosphorylamino acid antibody which is specific for saidphosphorylated amino acid, and which selectively binds to a moleculecomprising said phosphorylated amino acid; and

(iv) a high energy phosphate source

(b) measuring the FP, FQ, or FCS of said reporter following thecombination of step (a); and

(c) using the FP, FQ, or FCS measurement of step (b) to determine theactivity of said enzyme.

In one embodiment, the substrate is combined with the enzyme before theaddition of said reporter and said antibody. In another embodiment, thesubstrate, the reporter, and the antibody are combined with the enzymesimultaneously.

As described, the dephosphorylation of a substrate peptide or proteincan be also be measured by the competitive immunoassays of the presentinvention. Thus, in another embodiment, the invention provides a methodfor determining the dephosphorylating activity of an enzyme comprisingthe steps of:

(a) combining said enzyme with

(i) a reporter molecule comprising a fluorescent label and aphosphorylated amino acid, wherein said amino acid is selected from thegroup consisting of serine, threonine and tyrosine;

(ii) a substrate comprising the same phosphorylated amino acid as saidreporter, wherein said substrate is capable of being dephosphorylated atsaid amino acid by said enzyme; and

(iii) an antibody which selectively binds to a molecule comprising saidphosphorylated amino acid;

(b) measuring the FP, FQ, or FCS of said reporter following thecombination of step (a); and

(c) using the FP, FQ, or FCS measurement of step (b) to determine thedephosphorylating activity of the enzyme.

As in the kinase assay described above, the antibody and reportermolecule may be added after the phosphatase reaction has proceeded forsome time, in which case the remaining phosphorylated substrate willspecifically displace the reporter molecule from the antibody.Alternatively, the antibody and reporter molecule may be present attime=0, so that the phosphorylated substrate competes with the reporterfor the antibody, giving intermediate values of polarization and/orquenching. As is true for the kinase assay, the antibody and reportermolecule can only be present at the time that the enzyme is added whereneither the antibody nor the reporter are a substrate for the enzyme.

Because the methods of the invention utilize a competitive immunoassayto determine the amount of phosphorylated or dephosphorylated substrate(i.e., the amount of product) produced, the amount of phosphorylatedsubstrate required to displace the reporter from the antibody will varydepending upon the K_(d) of the phosphorylated substrate for theantibody and the K_(d) of the antibody for the reporter molecule. Thus,where the K_(d) of the phosphorylated substrate for the antibody is,e.g., 10-fold less than the K_(d) of the antibody for the reportermolecule, then an amount of phosphorylated substrate ten times higherthan the amount of reporter will be required for the phosphorylatedsubstrate to displace the reporter from the antibody. Where the K_(d) ofthe phosphorylated substrate for the antibody is approximately equal tothe K_(d) of the antibody for the reporter molecule, only 50% of themaximal signal can be achieved. In a highly preferred embodiment, theK_(d) of the phosphorylated substrate for the antibody will be less thanthe K_(d) of the antibody for the reporter molecule. In this situation,phosphorylation of the substrate will quantitatively displace thereporter from the antibody.

Another way of reducing the amount of substrate relates to the choice ofreporter molecule used in the methods of the invention. A suitablereporter molecule to be used in the methods of the invention is afluorescently-labeled molecule, preferably a peptide or protein,comprising a phosphorylated amino acid, wherein said amino acid isselected from the group consisting of serine, threonine, and tyrosine.Suitable reporter molecules for use in the methods of the presentinvention thus include fluorescently labeled phosphoserine,phosphothreonine or phosphotyrosine, as well as an appropriatelyfluorescently labeled peptide comprising a phosphorylamino acid (P-AA)selected from the group consisting of Ser, Thr and Tyr. Selection ofreporter wherein the K_(d) of the antibody for the reporter is higherthat the K_(d) of the phosphorylated substrate for the antibody willreduce the amount of substrate needed to displace the reporter from theantibody. Because anti-phosphorylamino acid antibodies may have a higheraffinity for a fluorescently labeled phosphorylamino acid than for afluorescently labeled peptide comprising the same phosphorylamino acid,in a preferred embodiment, such peptides are used as the reporter.

Production of peptides to be used as reporters may be accomplished byany one of a number of methods that are well known to those of ordinaryskill, such as by enzymatic cleavage, chemical synthesis, or expressionof a recombinantly produced peptide. The reporter peptide may besynthesized and then phosphorylated, or instead the phosphorylated aminoacid or amino acids may be incorporated into the peptide at the timethat the peptide is synthesized. Phosphorylamino acids for incorporationinto chemically synthesized peptides may be obtained from numerouscommercial sources, such as Sigma (St. Louis, Mo.). In preferredembodiment, labeling of the reporter is accomplished by including a Cysresidue in the sequence one to two residues away from thephosphorylatable amino acid.

Suitable fluorescent labels to be used in the methods of the inventioninclude any fluorophore that, upon the binding of the reporter moleculeby an anti-phosphorylamino acid antibody, undergoes a change detectableby FP, FQ, or FCS. Of course, in order to be used in the methods of theinvention, the label cannot interfere with recognition of the reporterby the anti-phosphorylamino acid antibody. Where the detection method tobe used is FP, the fluorescent label to be used may be any probe which,when combined with a molecule comprising a phosphorylated amino acid(P-AA) selected from the group consisting of Ser, Thr and Tyr to form areporter molecule, undergoes a change in fluorescence lifetime when thereporter molecule binds to a larger molecule (i.e., the antibody). Wherethe detection method to be used is FQ, the fluorescent label to be usedmay be any environment-sensitive probe which, when combined with amolecule comprising a phosphorylamino acid (P-AA) selected from thegroup consisting of Ser, Thr and Tyr to form a reporter molecule,undergoes a change in fluorescence intensity when the reporter moleculebinds to a larger molecule (i.e., the antibody). In FCS, the differencein the diffusion coefficients of two bound molecules (with the smallerof the two being fluorescently labeled) is observed in a very smallvolume. Thus, the smaller, labeled molecule will diffuse into theobserved volume faster in the unbound state than it will if bound to alarger molecule. For this technique, any probe that undergoes minimalphotobleaching can be used, with preference given to those with thehighest quantum yields.

Utilization of a FRET assay format results in a continuous recordingassay for either phosphatase or kinase activities. Where a FRET formatis to be used, the antibody is labeled with a suitable donor or acceptorfluorophore, while the substrate is labeled with the donor or acceptorfluorophore that complements the fluorophore labeling the antibody. Forexample, the substrate peptide can be labeled with a donor fluorophoresuch as fluorescein or Oregon green, while the antibody is labeled witha suitable fluorophore acceptor, such as rhodamine, with excitation atthe absorbance maximum of the donor, and emission observed at themaximum of either fluorophore. Thus, as the target amino acid of thelabeled substrate becomes phosphorylated, the labeled antibody will bindto the substrate, resulting in both the quenching of the donorfluorescence and the enhancement of the acceptor fluorescence.

An antibody suitable for use in the FP, FQ, FRET and FCS methods of theinvention is one that binds specifically to phosphoserine,phosphothreonine, or phosphotyrosine, and that produces changes ineither the intrinsic polarization or quenching of the emission intensityof the fluorosceinated version of the phosphorylamino acid to which itbinds. Furthermore, where the assay format used is FP, FQ, or FCS, thedifference between the K_(d) of the antibody for the phosphorylatedsubstrate and the K_(d) of the antibody for the reporter molecule willdictate the amount of phosphorylated substrate that must be present inorder for the phosphorylated substrate to displace the reporter from theantibody. Thus, where the K_(d) of the phosphorylated substrate for theantibody is, e.g., 10-fold less than the K_(d) of the antibody for thereporter molecule, then an amount of phosphorylated substrate ten timeshigher than the amount of reporter will be required for thephosphorylated substrate to displace the reporter from the antibody.Where the K_(d) of the phosphorylated substrate for the antibody isapproximately equal to the K_(d) of the antibody for the reportermolecule, only 50% of the maximal signal can be achieved. In a highlypreferred embodiment, the K_(d) of the phosphorylated substrate for theantibody will be less than the K_(d) of the antibody for the reportermolecule. In this situation, phosphorylation of the substrate willquantitatively displace the reporter from the antibody.

Because the methods of the invention are to be used to assay kinase andphosphatase activity, it is preferred that displacement of the reportermolecule from the antibody be accomplished with a relatively lowconcentration of phosphorylated substrate. As is described above, thismay be accomplished by using a reporter having a K_(d) for the antibodythat is higher than the K_(d) of the phosphorylated substrate for theantibody, and will allow detection of lower concentrations ofphosphorylated substrate. With proper selection of the reporter, theassay format can be FP, FQ, or FCS.

Anti-phosphorylamino acid antibodies may be obtained from numerouscommercial sources, including Sigma (St. Louis, Mo.), ICN Biomedicals(Costa Mesa, Calif.), Life Technologies (Gaithersburg, Md.),Transduction Labs, (Lexington, Ky.), Molecular Biology Laboratories(Nagoya, Japan), Upstate Biologicals (Lake Placid, N.Y.) and ZymedLaboratories (South San Francisco, Calif.). In addition,anti-phosphorylamino acid antibodies may be prepared as described inZoppini et al. (Eur. J. Lab. Med. 1(2):101-103 (1993)), and in thereferences cited therein. Determination of whether a certain anti-P-AAantibody produces changes in either the intrinsic polarization orquenching of the emission intensity of the F-P-AA may be made accordingto the method described in Example 1.

As used herein, the term “antibody” includes monoclonal antibodies,polyclonal antibodies, single chain antibodies, and ligand-bindingfragments of antibodies, such as Fab and F(ab′)₂. Various proceduresknown in the art may be used to produce such antibodies and fragments.For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures may be used, suchas the hybridoma technique of Kohler and Milstein (Nature, 256:495-497(1975)). Techniques for the production of single-chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies tophosphoserine, phosphothreonine, or phosphotyrosine.

As may be seen from the above discussion, the assay format gives theuser a great deal of latitude for tailoring reagents and reactionconditions to meet the requirements of a specific kinase or phosphatase.

Changes in the fluorescent properties of the reporter may be measuredusing any technique capable of detecting a change in the fluorescentproperties of a molecule. Such methods include, but are not limited to,fluorescence polarization, fluorescence quenching, fluorescenceresonance energy transfer, and fluorescence correlation spectroscopy.

Fluorescence polarization: Fluorescence polarization (FP), in contrastto intensity measurements, can readily be used in the development oftrue, homogeneous, solution assays, or for real time, continuousrecording assays, as the method can generate a direct quantitation ofthe ratio of bound/free ligand. This is extremely cost effective interms of simplicity of operations and the types of screening laboratoryware required for the assay. Since polarization is independent of thefluorescence intensity, this technique can be used in the presence ofcolored compounds which may quench the emission.

When a fluorescent molecule is excited by polarized light, its emissionis also polarized. The degree of polarization is dependent upon theviscosity of the solution, the rotational correlation time of thefluorophore (φ), and the temperature of the reaction mixture.Mathematically, the steady-state polarization (P) is expressed as$\begin{matrix}{P = \frac{I_{1} - I_{\bot}}{I_{1} + I_{\bot}}} & (2)\end{matrix}$

which is the difference between the intensities (I) of the parallel andperpendicular components of the polarized emission divided by their sum.More recently, the fluorescence anisotropy (r), which is dependent onthe extent of rotational motion during the lifetime of the excitedstate, is being used because theoretical expressions are simpler whenexpressed in terms of this parameter rather than P. P is related to r bythe expression $\begin{matrix}{P = {\frac{3r}{2 + r}.}} & (3)\end{matrix}$

Since P, unlike r, is not completely linear with fraction bound, it ismore beneficial to use r for the calculations, especially if the quantumyields of the free (q_(F)) and bound (q_(B)) species are not equal. Thecorrected fraction bound in this case is given by $\begin{matrix}{{FB} = \frac{r - r_{F}}{{\left( {r_{B} - r} \right)R} + r - r_{F}}} & (4)\end{matrix}$

where r_(F) and r_(B) are the anisotropies of the totally free andtotally bound species respectively, r is the anisotropy of theexperimentally observed bound species, and R=q_(B)/q_(F). Titration ofthe fluorescent component with the nonfluorescent component yields datawhich is readily analyzable by various forms of the Langmuir bindingisotherm or the stoichiometric binding equation if the binding isspecific.

The requirements for a polarization assay are: (1) an assay component(the one with the lowest molecular weight) must be labelled with a longlifetime fluorescent probe and retain its ability to bind to the othercomponent(s), (2) there must be a sufficient difference between themolecular weight of the labelled component and the nonlabeled one suchthat the probe senses a significant volume change upon binding, (3) theprobe must have a relatively high quantum yield so that its fluorescenceat low concentrations is significantly greater than background (4) thetemperature and viscosity of the reaction mixture must be strictlycontrolled, and (5) the polarization should increase in a dose-dependentsaturable manner. For use in plate-reader format, the fluorophore usedshould be a bright visible probe such as, e.g., fluorescein orrhodamine. Thus, where the detection method to be used is fluorescencepolarization, the fluorescent label to be used may be anyenvironment-sensitive probe whose fluorescence lifetime changes uponbinding to a larger molecule.

The changes observed in the anisotropy free and bound ligands are afunction of their individual rotational correlation times. Thus, inorder to obtain a good dynamic range for the assay, the rotationalcorrelation time of the labeled antigen should be shorter than thelifetime of the fluorescent tag. Since most visible probes havelifetimes ≦10 ns, the polarization assay is limited generally to thebinding of small labelled ligands to large unlabelled targets. Thus itis generally not possible to study by polarization methods theinteraction of two large macromolecules.

Fluorescence Quenching: HTS assays that utilize the intensity of thefluorophore as a readout usually require the separation of the free andbound species unless an environment-sensitive fluorophore is utilized.However, there are very few probes of this nature which both absorb andemit in the visible range, and as such, are not useful for HTS. In somecases, however, binding of the nonfluorescent component to the labelledligand may result in a concentration-dependent quenching or enhancementof the fluorescence which may occur due to the presence of quenchinggroups in the unlabelled component or some chemistry of the binding sitewhich affects the fluorescence emission of the fluorophore. Thus theconcentration-dependent decrease (or increase) in the fluorescenceemission may be used to quantitate binding. The advantage of thisreadout is that a simple fluorescence plate reader may be used withoutpolarizers. The usefulness of this assay may be limited where coloredcompounds which also quench the fluorophore are present in the assaymixture. Thus, where the detection method to be used is fluorescencequenching, the fluorescent label to be used may be anyenvironment-sensitive probe whose fluorescence intensity changes uponbinding to a larger molecule.

Fluorescence Resonance Energy Transfer: Fluorescence resonance energytransfer (FRET) is the transfer of the excited state energy from a donor(D) to an acceptor (A), and occurs only when the emission spectrum ofthe donor (D) fluorophore overlaps the absorption spectrum of theacceptor (A) fluorophore. Thus, by exciting at the absorption maximum ofthe donor and monitoring the emission at the long wavelength side of theacceptor fluorophore, it is possible to monitor only D and A moleculesthat are bound and reside within a certain distance, r. Thus one canmonitor either the quenching of D or enhanced emission of A. Thetransfer rate, k_(T) in sec⁻¹ is mathematically defined as

k _(T)=(r ⁻⁶ Jκ ² n ⁻⁴λ_(d))×8.71×10²³  (5)

where r is the D-A distance in angstroms, J is the D-A overlap integral,κ² is the orientation factor, n is the refractive index of the media,and λ_(d) is the emissive rate of the donor. The overlap integral, J, isexpressed on the wavelength scale by $\begin{matrix}{J = {\int_{0}^{\infty}{{F_{d}(\lambda)}{\varepsilon_{a}(\lambda)}\lambda^{4}\quad {\lambda}}}} & (6)\end{matrix}$

where its units are M⁻¹cm³, F_(d) is the corrected fluorescenceintensity of the donor as a function of wavelength λ, and ε_(a) is theextinction coefficient of the acceptor in M⁻¹cm⁻¹.

Constant terms in equation 4 are generally combined to define theForster critical distance, R_(o), which is the distance in angstroms atwhich 50% transfer occurs. By substitution then, R_(o) can be defined interms of the overlap integral, J, in angstroms, as

R _(o)=9.79×10³(κ² n ⁻⁴φ_(d) J)^(1/6)  (7)

with φ_(d) being the quantum yield of the donor.

R_(o) and r are related to the transfer efficiency, E, by$\begin{matrix}{E = \frac{R_{o}^{6}}{R_{o}^{6} + r^{6}}} & (8)\end{matrix}$

which determines the practical distance by which D and A can beseparated to obtain a usable signal.

From these equations it is easy to see that, for high sensitivity, it isimportant to choose D-A pairs which have high quantum yields, high Jvalues, and high R_(o) values. For example, R_(o) for thefluorescein/rhodamine pair is about 55 Å. Large values of R_(o) arenecessary to achieve a measurable signal when molecules containing D andA bind to each other. In practice it is common to use twice as manyacceptor as donor molecules in the reaction mixture if the emission of Ais to be used as the readout. Thus, binding of, for example,macromolecule I labelled with D, to macromolecule II labelled with A,can be detected by the emission of A when excited at the absorption ofD. Again, for a competitive process, the concentration increase in Afluorescence must occur in a hyperbolic, saturable manner. The FRETassay is thus especially desirable for monitoring the binding of twolarge macromolecules where FP techniques are not particularly useful.

In view of the guidelines provided above and in the Examples, below, theskilled practitioner will be able to choose the detection technique bestsuited for the particular assay being performed.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1

Materials and Methods

(a) Reagents: Antibodies to phosphoryltyrosine were obtained from ICNBiomedicals, Inc., Costa Mesa, Calif., (monoclonal antibody clone PY20,Catalog Number 69-137), Life Technologies, Gaithersberg, Md.,(monoclonal antibody clone 6G9, Catalog Number 13160-011), MolecularBiology Laboratories, Nagoya, Japan (monoclonal antibody clone 6D12;Catalog Number MH-11-3), Sigma, St. Louis Mo., (monoclonal antibodyclone PT-66, Catalog Number B-1531), Transduction Labs, Lexington, Ky.(monoclonal antibody clone PY20, Catalog Number P11120; altered Fab ofPY20 produced in E. coli, Catalog Number E120H), Upstate BiologicalsInc., Lake Placid N.Y., (monoclonal antibody clone 4G10, Catalog Number05-321), and Zymed Laboratories, Inc., South San Francisco, Calif.,(monoclonal antibodies, clone PY-7E1, Catalog Number 13-5900; clonePY-IB2, Catalog Number 13-6300; clone PY20, Catalog Number 03-7700;clone Z027, Catalog Number 03-5800; and polyclonal antibodies CatalogNumber 61-5800 and 61-8300).

Antibodies to phosphorylthreonine were obtained from Sigma (monoclonalantibody clone PTR-8, Catalog Number B-7661) and Zymed (monoclonalantibody, clone PT-5H5, Catalog Number 13-9200; polyclonal antibody,Catalog Number 61-8200). Antibodies to phosphorylserine were obtainedfrom Sigma (monoclonal antibody clone PSR-45, Catalog Number B-7911) andZymed (polyclonal antibody, Catalog Number 61-8100). The structures ofall chemicals used are shown in FIG. 1.

(b) Synthesis of amino-fluorescinated phosphorylamino acids: Theamino-fluoresceinated, phosphorylamino acids (i.e.—serine, threonine andtyrosine) were synthesized by reacting the phosphorylamino acids withfluorescein isothiocyanate (FITC) (Pierce Chemical Company) in 0.1 MNaHCO₃ of pH 9. The FITC used was a mixture of the two isomers shown inFIG. 1. Purification was performed using a C4 Reverse Phase column (2.5mm×25 cm), a flow-rate of 0.5 ml/min, and a 15 minute gradient from 0.01N HCl to 100% CH₃CN. The fluoresceinated phosphorylamino acids wereidentified by mass spectral analysis. For these compounds, a molarextinction coefficient of 77,000 was assumed in pH 8 buffer with a 1 cmpathlength.

(c) Fluorescence Measurements: Ratiometric fluorescence measurementswere made using an ISS K2 spectrofluorometer equipped with GlansThompson quartz polarizers. The temperature of the reaction cuvette wasmaintained at 25.0° C. by a Lauda RM6 circulating bath. The fluorescenceof the labelled phoshorylamino acids was measured with excitation at 485nm and emission was observed through an Omega 530±15 nm bandpass filter.

Antibody binding to the fluoresceinated phosphorylamino acids wasmonitored by adding successive small volumes of the antibody stocksolution to a cuvette containing a fixed amount of fluoresceinatedphosphorylamino acid in two milliliters of pH 7.4 buffer (25 mM Tris and25 mM NaCl adjusted to pH 7.4 with HCl). The fluorescence polarization,anisotropy, and the intensity—the sum of vertically and horizontallypolarized emissions—were recorded one minute after each addition. Thedilution produced by the addition of the antibody necessitated a slightcorrection for both the concentrations and the intensity readings butnot for the polarization since the latter is independent of thefluorophore concentration. Displacement of the fluorophore from theantibody by competing nonfluorescent ligands was measured by using fixedamounts of antibody and fluorophore in two milliliters of pH 7.4 bufferand adding successive small volumes of the competitor stock solution.Again, the displacement was monitored one minute after each addition bythe changes in fluorescence intensity, polarization, and anisotropy

Data Analysis: Data analysis was performed as follows. It is assumedthat the binding of the fluorophore ligand, L, and the competinginhibitor, I, to the antibody, A, are rapid, simple thermodynamicequilibria, according to the scheme:

with K_(l)=L·A/C and K_(i)=I·A/X.

Case I: Displacement with A_(o)>>L_(o): For the displacementexperiments—where both L_(o) and A_(o) are constant and I_(o)increases—the analysis of the data is greatly simplified when it ispossible to use experimental conditions where I_(o)>>A_(o) andA_(o)>>L_(o). Under these conditions, L=L_(o)−C; I=I_(o);X=A·I_(o)/K_(i); A_(o)=A+X A(1+I_(o)/K_(i)). ThereforeA=A_(o)/(1+I_(o)/K_(i)) and $\begin{matrix}{K_{1} = \frac{\left( {L_{o} - C} \right)A_{o}}{C \cdot \left( {1 + \frac{I_{o}}{K_{i}}} \right)}} & (3)\end{matrix}$

Upon solving (3) for C we obtain: $\begin{matrix}{C = {\frac{C_{u}}{1 + \frac{I_{o}}{K_{i}^{app}}}.}} & (4)\end{matrix}$

in which we define the concentration of the fluorophore/antibody complexin the absence of inhibitor as C_(u)=L_(o)/(1+K_(l)/A_(o)) and theapparent inhibition constant as K_(i) ^(app)=K_(i)·(1+A_(o)/K_(l)). Inthese experiments one can directly determine only K_(i) ^(app). Thevalue of K_(i) can be calculated only if L_(o) and K_(l) are known.

Case IIa: Binding: The analysis of the direct binding of a constantconcentration of fluorophore, L_(o), to a variable concentration ofantibody, A_(o), of comparable magnitude requires solving a quadraticequation. By substituting L and A from the stoichiometric equationsL_(o)=L+C and A_(o)=A +C into the definition of K_(l) we obtain:$\begin{matrix}{K_{1} = \frac{\left( {L_{o} - C} \right) \cdot \left( {A_{o} - C} \right)}{C}} & (5)\end{matrix}$

which yields the quadratic equation:

 C ² −C·(A_(o) +L _(o) +K _(l))+L _(o) A _(o)=0  (6)

The solution of (6) is $\begin{matrix}{C = {\frac{1}{2} \cdot \left( {A_{o} + L_{o} + K_{1} - \sqrt{\left( {A_{o} + L_{o} + K_{1}} \right)^{2} - {4 \cdot A_{o} \cdot L_{o}}}} \right)}} & (7)\end{matrix}$

Case IIb: Displacement: The usual conditions for the displacementexperiments are L_(o) and A_(o) constant but of comparable magnitudewith increasing I_(o) and I_(o)>>A_(o). Under these conditions,L=L_(o)−C; I=I_(o); and X=A·I_(o)/K_(i). ThereforeA_(o)=A+C+X=C+A·(1I_(o)/K_(i)) so that $\begin{matrix}{A = \frac{A_{o} - C}{1 + \frac{I_{o}}{K_{i}}}} & (8) \\{{{{and}\quad K_{1}C} = {{L \cdot A} = \frac{\left( {L_{o} - C} \right) \cdot \left( {A_{o} - C} \right)}{1 + \frac{I_{o}}{K_{i}}}}}{{{Solving}{\quad \quad}(9)}\quad {for}\quad C\quad {yields}}} & (9) \\{C = {\frac{1}{2} \cdot \left( {A_{o} + L_{o} + {K_{1} \cdot \left( {1 + \frac{I_{o}}{K_{i}}} \right)} - \sqrt{\left\lbrack {A_{o} + L_{o} + {K_{1} \cdot \left( {1 + \frac{I_{o}}{K_{i}}} \right)}} \right\rbrack^{2} - {4 \cdot A_{o} \cdot L_{o}}}} \right)}} & (10)\end{matrix}$

Fluorescence Intensity: The fluorescence intensity, F, of a mixture of Land C is

F=Q _(L) ·L+Q _(C) ·C=Q _(L) ·L _(o)+(Q _(L) −Q _(C))·C  (11)

where Q_(L) is the molar emissivity of the free fluorophore and Q_(C)that of the bound one. Substituting into this equation C from Eq 7yields the quadratic used to analyze the fluorescence intensity bindingdata obtained under Case IIa.

Similarly, substituting the expression for C from Eq 4 or from Eq 10yields the expression for analyzing the fluorescence displacement dataunder Cases I or IIb, respectively.

Fluorescence Polarization and Anisotropy: The analysis of the changes influorescence intensity upon binding to the antibody are relativelysimple because the changes are a linear function of the composition ofthe L+C mixture. As pointed out previously (7), this is not the case ingeneral when changes in fluorescence polarization or anisotropy aremeasured. Indeed, if the exciting electric vector is vertical, then thepolarization of a solution is a function of the emitted intensitieshorizontally polarized, h, and vertically polarized, v, according to thedefinition: $\begin{matrix}{p = \frac{v - h}{v + h}} & (12)\end{matrix}$

The total fluorescence intensity is F=h+v=Q·M, where Q is the molaremissivity of the fluorophore at concentration M. Solving Eq 12 for vyields v=h·(1+p)/(1−p). Thus: $\begin{matrix}{{Q \cdot M} = {h \cdot \left( {1 + \frac{1 + p}{1 - p}} \right)}} & (13)\end{matrix}$

and it follows that $\begin{matrix}\begin{matrix}{h = \frac{Q \cdot {M\left( {1 - p} \right)}}{2}} \\{with}\end{matrix} & (14) \\{v = \frac{Q \cdot {M\left( {1 + p} \right)}}{2}} & (15)\end{matrix}$

For a mixture of L and C, the polarizations calculated from the sum ofthe vertically and horizontally polarized emission intensities are:$\begin{matrix}{p = \frac{v_{L} + v_{C} - h_{L} - h_{C}}{v_{L} + v_{C} + h_{L} + h_{C}}} & (16)\end{matrix}$

Substituting h_(L)=Q_(L)L(1−P_(L))/2, h_(C)=Q_(C)C(1−p_(C))/2,v_(L)=Q_(L)L(1+p_(L))/2, and v_(c)=Q_(C)C(1+p_(C))/2, where p_(C) andp_(L) are the polarization values for the bound ligand and the freeligand, respectively, and then solving for C/L yields, as reportedearlier (7): $\begin{matrix}{\frac{C}{L} = {\frac{C}{L_{o} - C} = {\frac{Q_{L}}{Q_{C}}\frac{p - p_{L}}{p_{C} - p}}}} & (17)\end{matrix}$

Similarly, from the definition of the fluorescence anisotropy, a,:$\begin{matrix}{a = \frac{v - h}{v + {2h}}} & (18)\end{matrix}$

we derive: $\begin{matrix}{\frac{C}{L} = {\frac{C}{L_{o} - C} = {\frac{Q_{L}}{Q_{C}}\frac{2 + a_{C}}{2 + a_{L}}\frac{a - a_{L}}{a_{C} - a}}}} & (19)\end{matrix}$

where a_(C) and a_(L) are the fluorescence anisotropies for the boundligand and the free ligand, respectively.

Eq 17 can be solved for p as a function of C: $\begin{matrix}{p = \frac{{p_{L}L_{o}} + {C\left\lbrack {{\frac{Q_{C}}{Q_{L}}p_{C}} - p_{L}} \right\rbrack}}{L_{o} + {C\left\lbrack {\frac{Q_{C}}{Q_{L}} - 1} \right\rbrack}}} & (20)\end{matrix}$

Of course, when the molar emissivity does not change upon binding, i.e.when Q_(C)/Q_(L)=1, then p becomes a linear function of C, in fullanalogy with the fluorescence intensity as expressed in Eq 11.

When Q_(C)/Q_(L)≠1 then substituting into Eq 20 the value of C from theappropriate equation (Eq 4, Eq 7, or Eq 10) allows one to determineK_(l) and K^(app) _(i) or K_(i) by nonlinear least squares analysis ofthe polarization data. Depending on the experimental conditions used,such an analysis may also yield the best-fit values for p_(L), p_(C),Q_(L), and Q_(C).

Results

The majority of the anti-phosphorylamino acid antibodies available fromsuppliers were evaluated, looking for those that produced changes ineither the intrinsic polarization or quenching of the emission intensityof the fluoresceinated phosphorylamino acids. From the results of thissurvey, three classes of antibodies were identified forphosphoryltyrosine, based on the types of fluorescent signal produced bythe antibody: (I) those giving a large polarization change withoutsignificant effect on the fluorescence emission of the fluorescentphosphorylamino acid, (II) those producing polarization changes andquenching of the emission, and (III) those yielding little or no changein either parameter, or produced noisy, nonreproducible data.

Due to sensitivity considerations, titrations of the fluorescentphosphorylamino acids with the antibodies were performed at comparableconcentrations of A_(o) and L_(o). Under these conditions, one must usethe quadratic form of the binding equation (Equation 7) in order todetermine with precision the K_(d) of the ligand for the antibody andthe stoichiometry of the reaction. The concentration of L_(o) wascalculated from the extinction coefficient of the fluorophore. However,the concentration of the stock solution of the antibody is given inunits of protein concentration/ml, not active antibody which has twobinding sites/molecule.

The antibody concentrations were first calculated using the proteinconcentrations supplied by the manufacturer, assuming a molecular weightof 160,000. [1]. Calculations done in this manner do not take intoconsideration the bidentate nature of the antibody. Thus, thesite-concentration or normality of the antibody should be twice theconcentration calculated as described above. Calculation of antibodyconcentrations based on protein quantitation would exceed the actualantibody concentration, due to the presence of extraneous contaminatingproteins and/or inactive antibody. For these reasons, in the titrationcurves, antibody concentrations are given in terms of the volume ofantibody added, and the actual K_(d)s of the fluorescent phosphorylaminoacids are calculated from the fitted stoichiometry, which hasconcentration units of molar sites (i.e., normality). Comparison of thedetermined stoichiometry with concentrations calculated from proteinconcentrations can then be used to assess the purity and activity ofantibodies from different manufacturers.

Examples of experimental results using Type I antibodies (as describedabove) are shown in FIG. 2. Addition of any of the Sigmaanti-phosphorylamino acid antibodies to the correspondingfluoresceinated phosphorylamino acid produced a steep,concentration-dependent increase in the polarization of the fluorescencewhich was saturable at higher antibody concentrations. The maximalincrease in polarization at saturating antibody concentrations wasapproximately 9-fold for antibody binding to labelled(i.e.—fluoresceinated) phosphorylamino acids with an excellentsignal-to-noise ratio. The Sigma antibodies are thus Type I antibodies(FP change only). The data sets were analyzed using Equation 7 inconjunction with a nonlinear least squares fitting program and areconsistent with this model as evidenced by the agreement between theexperimental data points and the theoretical curves. The dissociationconstant for all the Sigma antibodies, calculated from the fits to theexperimental data, are given in Table 1 in units of antibody normality.All three fluoresceinated phosphorylamino acids had a high picomolar tolow nanomolar affinities for their corresponding antibodies.

The antibodies were then individually evaluated for theircross-reactivity with the opposite fluoresceinated phosphorylaminoacids, and the non-phosphorylated amino acids themselves by competitivedisplacement immunoassay. None of the antibodies were found to crossreact with the other phosphorylamino or amino acids at levels 10-20 foldabove those used in the titration experiments. The data for theanti-phosphoserine and anti-phosphothreonine antibodies are discussedmore extensively below.

Three anti-phosphotyrosine antibodies which gave both significantincreases in the fluorescence polarization and decreases in the emissionintensity when bound to P-Tyr-F were also identified (Type IIantibodies). Two antibodies, from ICN and MBL, produced the largestdecreases in the fluorescence of P-Tyr-F upon binding (Table 1; asQ_(f)/Q_(b) increases so does the dynamic range).

Analysis of the data in FIGS. 3 and 4 using Equations 7 and 20 resultedin fits that were consistent with the experimental points, and there wasgood agreement between the K_(d)s (low nM) calculated from both thepolarization and the intensity readings. Thus, kinase and phosphataseassays utilizing the ICN or MBL antibodies may be performed using eitherthe fluorescence polarization increase or emission quenching as ameasure of antibody binding to labeled phosphorylamino acids in thepresence of unlabeled phosphorylated reaction products. The cause of theemission quenching by the ICN and MBL antibodies may involve hydrogenbonding of the fluorescent ligand, the presence of tryptophan residuesin or near the binding site, or electrostatic interactions.

Other antibodies were found to produce small, nonrobust fluorescencechanges upon binding labelled ligand (Table 1), and were considered notto be useful for the kinase assays. Still other antibodies testedproduced insignificant or inconsistent changes in either the emissionintensity or polarization, and were deemed not usable for either kinaseor phosphatase activity detection (Type III antibodies). Interestingly,the superior antibodies (those that gave the most robust signals withthe least scatter) had normalities calculated from the fits to thequadratic equation that were in good agreement with concentrationssupplied by the manufacturer. In contrast, for the less robustantibodies, there was a great disparity between the stoichiometry andprotein concentration calculations, indicating the presence of eithercontaminating proteins and/or inactive antibody.

Antibodies for phosphoserine and phosphothreonine came from two sources,Sigma (St. Louis, Mo.) and Zymed (CA). Only the Sigma antibodies werefound to produce a signal of significant magnitude that would allow, forkinase and phosphatase assays, accurate quantitation of unlabelledphosphoserine and phosphothreonine (Table 2). The results fromexperiments with the Sigma antibodies were calculated by nonlinear leastsquares analysis using equation 7, are shown in FIG. 2. None of theSigma antibodies produced quenching of the emission intensities, butboth P-Ser-F and P-Thr-F had high affinity for their respectiveantibodies as measured by FP. Although binding of the fluorescentligands to the Zymed antibodies gave both polarization and intensitychanges, they were accompanied by significant scatter and a less thandesirable dynamic range to be useful for monitoring either kinase orphosphatase activity.

In order to determine if kinase reactions can be followed by competitionof the phosphorylated substrate with labelled phosphorylamino acid, thespecificity of the binding by the labeled phoshorylamino acids wasdemonstrated by co-competition with their unlabeled counterparts for theantibody-bound fluorescent complex. The competition between the labeledand unlabeled phosphorylamino acids for the antibodies was firstdetermined. The amounts of antibody and fluorescent phosphorylamino acidwere held constant (close to the K_(d)) in these experiments, and thedecrease in polarization or increase in emission resulting from theaddition of the unlabelled phosphorylamino acid was monitored as afunction of its concentration. The experimental data were analyzed byfixing the concentrations of L, K_(l), and Q_(L) using Equation 10 inconjunction with Equation 20 (when polarization with a quenchingantibody was measured), or Equation 10 only when the increasedpolarization was not accompanied by quenching. The results of theseexperiments are shown in FIGS. 4 (Sigma antibodies) and 5 (MBLantibody). Fitting in the manner described above yielded excellent fitsto the experimental data. The values for the corrected dissociationconstants of the phosphorylamino acids are given in Table 3. The K_(i)sfor phosphoryltyrosine, phosphorylserine, and phosphorylthreonine wereconsiderably higher than those for the fluoresceinated ligands. Goodagreement between K_(i)s calculated from the polarization and quenchingdata were determined for the MBL antibody. These experiments show thatkinase and phosphatase activities can be measured with precision usingeither FP or quenching data.

In order to calculate, by competition with labeled ligand, the amount ofphosphorylated or dephosphorylated substrates produced during the courseof the kinase or phosphatase reactions, it is imperative to know thedissociation constant of the phosphorylated substrate for the particularantibody. The affinities of two phosphorylated peptides which aresubstrates for the JAK-2 tyrosine kinase were measured by displacementof fluoresceinated phosphoryltyrosine from both the corresponding Sigmaand MBL antibodies. The structure of the first JAK-2 substrate,JAK-2(a), is:

The formula of the second JAK-2 substrate, JAK-2(b) is:

Both phosphorylated peptides displaced P-Tyr-F from the antibodies in adose-dependent manner, whether measured by FP only (Sigma) orsimultaneously using quenching or fluorescence polarization (MBL). Thedata were analyzed by nonlinear least squares analysis using equation 7,and the results are shown in FIGS. 6 and 7. The K_(i)s calculted fromthe fits are given in Table 3. The K_(i)s of the two phosphorylatedJAK-2 kinase substrates for Sigma antibody were one and one-half ordersof magnitude higher than those of the labelled ligands. Similar resultswere obtained for displacement from the MBL antibody as shown in FIG. 7,and the calculated K_(i)s for this antibody from both FP and quenchingmeasurements were in good agreement. Thus, using the fluoresceinatedphosphorylamino acids in a competition reaction to measure kinase andphosphatase activities requires that the concentrations of addedfluoresceinated ligand should be comparable to K_(d) and the amount of Aup to 10×L. Under these degenerate conditions, the data can then beanalyzed by Equation 4.

Discussion

Two superior anti-phosphotyrosine antibodies were identified thatproduced changes in the fluorescence polarization of a bound ligand orquenching of its emission intensity, and would enable quantitation ofthe activities of any phosphatase or kinase. Kinase phosphorylation of asubstrate peptide or protein was monitored by specific displacement of afluorescent phosphorylamino acid or a fluorescent phosphorylated peptidefrom an antibody by the reaction product from the kinase assay (seereaction scheme below). A proposed assay format for the fluorescentkinase assay is given below.

In this scheme, the kinase reaction proceeds for a period of time, isstopped by the addition of EDTA, and both antibody and fluoresceinatedphosphorylamino acid added. Alternatively the antibody and labeledligand could be present at time=0. The phosphorylated substrate wouldcompete with the fluoresceinated phosphorylamino acid for the antibody,giving intermediate values of polarization and/or quenching. Phosphataseactivity can also be monitored using this immunoassay. Severalfluorescent substrates have previously been used to kinetically followphosphatase activity. These include phosphotyrosine (Z. Y. Zhang et al.,Anal. Biochem 211:7-15 (1993); B. Galvan et al, Clin. Biochem.29:125-131 (1996)), 2-methoxybenzoyl phosphate (P. Paoli et al.,Experientia 51:57-62 (1995)), europium-labeled antibody (D. Worm,Diabetologia 39:142-148 (1996)); terbium chelates (T. K. Christopoulos,Anal. Chem. 64:342-346 (1992)), and fluorescein diphosphate (E. Tolsa etal., J. Immunol. Methods 192:165-172 (1996)), with fluoresceindiphosphate being the most sensitive and amenable to HTS.

The evaluated antibodies demonstrated high affinities for thefluoresceinated phosphoryl amino acids. Therefore, a significantconcentration of phosphorylated peptide would be required to displacethe labeled ligand. This is detriment for kinase assays. However, thisproblem can be obviated by selecting three generic peptides, one foreach of the three amino acids, that is phosphorylated and fluorescentlylabeled. Peptides having reduced affinity for the antibodies will allowdetection of lower concentrations of phosphorylated product from thekinase reactions. With proper selection of the peptide and itsfluorescent labeling, the assay format can be FP, FRET, FQ, or FCS.

Two anti-phosphoryltyrosine antibodies (Sigma, St. Louis, Mo., and MBLInternational Corp., Watertown, Mass.) were identified that producedrobust fluorescence signals upon binding a labeled phosphoryltyrosine orphosphoryltyrosine peptide. The MBL antibody not only yielded a largepolarization change, but also significantly quenched (i.e., reduced by50%) the probe fluorescence. By using an antibody-reporter pair which,upon binding to each other, exhibit a change in both FP and FQ, thekinase assay described above can be performed in any fluorescent platereader, not just those with polarization capabilities. The use ofpolarization as a readout is desirable since this parameter isindependent of the fluorescence emission intensity (Leavine, L. M., etal., Anal. Biochem. 247:83-88 (1997)) and is thus not subject to theoptical artifacts imparted by colored compounds which may quench thefluorescence emission of the label. Several otheranti-phosphoryltyrosine antibodies from other suppliers were also tested(results not shown but the suppliers are listed in Materials andMethods) and found to produce low or inconsistent fluorescence changesupon binding the fluorescent ligand.

Four antibodies against phosphoserine and phosphothreonine (obtainedfrom Sigma and Zymed) were tested. The Sigma antibodies againstphosphoserine and phosphothreonine produced fluorescence polarizationchanges of sufficiently large dynamic range that the antibodies caneasily be used to measure the activities of specific kinases.

All three fluorescein labelled phosphorylamino acids had higheraffinities for their specific antibodies than their unlabeledcounterparts, presumably due to their relatively small size and theincreased hydrophobicity contributed by the fluorescein moiety. Due tothe high affinity of the antibodies for the labeled phosphorylaminoacids, a significant amount of product must be generated by kinaseactivity before displacement can occur. However, any labeledphosphorylated peptide or small protein, which would have lower affinityfor the antibody could instead be used as the competitive ligand,including fluorescein-modified phosphorylated JAK-2 substrate peptides.Thus, the assay format gives the user a great deal of latitude fortailoring reagents and reaction conditions to meet the requirements of aspecific kinase or phosphatase.

Example 2

Identification of Peptides having a Decreased Affinity for Antibody

Materials and Methods

Fluorescent Kinase Assay

As is discussed above, the displacement of reporter molecule byphosphorylated substrate in the competitive immunoassays of theinvention will occur at lower concentrations of phosphorylatedsubstrate, and thus most amenable to HTS, where the K_(d) of theantibody for the phosphorylated substrate is less than or equal to theK_(d) of the antibody for the reporter molecule. This may beaccomplished by obtaining a small phosphorylated labeled peptide whichhas a lower binding affinity toward its antibody than the correspondingfluorescent phosphorylated amino acid (between about 0.5 nM and 1 nM forfluoresceinated phosphotyrosine or phosphoserine). Appropriate peptidesmay have between about 3 and 50 amino acid residues, preferably betweenabout 3 and 25 residues, more preferably between about 3 and 15residues, and most preferably between about 4 and 10 amino acidresidues. Such peptides may be chemically synthesized, may be the resultof enzymatic or chemical cleavage of a larger peptide or protein, or maybe produced recombinantly. The peptides are then labeled and purified,and their affinities toward their corresponding antibodies are measured.Adjustments to the length and sequence of the peptides can be made tothe peptides if this is deemed necessary as a result of their K_(d) forantibody. Pentapeptides have been prepared which contain phosphorylatedamino acids with a free cysteine available for labeling with afluorophore. Peptides having the desired affinity (between about 50 nMand 100 nM) are then tested in the assay system for detecting thephosphorylation of substrate by kinases. As the assay is only measuringproduct, it will be possible to identify the actual amino acid of asubstrate that is being phosphorylated by a kinase using cocompetitionexperiments. For example, a peptide substrate that has beenphosphorylated by a kinase can be used to displace reporter peptidescontaining individual phosphorylated amino acids (i.e., either Ser, Thror Tyr) from their respective antibodies.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the invention.

The entire disclosure of all publications cited herein are herebyincorporated by reference.

TABLE 1 Affinities of Anti-phosphoryltyrosine Antibodies forFluoresceinated Phosphoryltyrosine Fluorescence PolarizationFluorescence Quenching K_(d), nM Sites Antibody K_(d), nM sites (p)¹ Δp¹(Φ)² Q_(f)/Q_(b) ³ Sigma 1.9 ± 0.3 x9 — — Gibco BRL 9.8 ± 7.3 x6 — — ICN3.8 ± 1.0 x6 5.1 ± 1.6 1.55 UBI bad data x5 81.8 ± 52.1 1.55 MBL 4.7 ±1.1 x9 3.9 ± 0.3 2.05 Zymed rabbit 6.6 ± 1.3 x8 1.7 ± 0.7 1.28polyclonal Zymed PY poor signal x2 4.2 ± 2.7 1.1  Plus ¹Directpolarization measurements ²Φ is the decreased Quantum Yield used tocalculate the K_(d) ³Ratio of the Quantum Yields of free and boundprobe. Used to correct polarization measurements as described in DataAnalysis.

TABLE 2 Affinities of Anti-phosphorylserine and Anti-phosphorylthreonineAntibodies for Fluoresceinated Phosphorylamino Acids FluorescencePolarization Antibody K_(d), nM Sites (p) Δp Sigma Anti P-Ser 0.30 ±0.03 x9 Zymed Anti P-Ser ≈8.13* x2.5 Sigma Anti P-Thr 1.055 ± 0.9 x5.1Zymed Anti P-thr 58.1 ± 10.1 x5 *Signal too noisy for accuratequantitation

TABLE 3 Affinities of Unlabelled Phosphorylamino Acids andPhosphorylated JAK-2 Substrates for their Respective Antibodies¹Phosphoryl Sigma Monoclonal MBL Monoclonal Ligand K_(d), nM (p) K_(d),nM Φ K_(d), nM (p) K_(d), nM Φ Phosphoryltyrosine 11.9 ± 2.5 —  5.5 ±1.0 5.4 ± 0.5 Phosphorylserine 16.8 ± 3.1 — — — Phosphorylthreonine 44.1± 6.0 — — — JAK-2(a) 15.4 ± 1.6 — 8.1 ± 1.8 11.6 ± 4.1 JAK-2(b) 49.5 ±3.6 — 12.1 ± 1.1   8.1 ± 0.9 ¹K_(d)s are calculated for nM sites (i.e. -normality)

What is claimed is:
 1. An article of manufacture comprising, packagedtogether: (i) a reporter molecule comprising a flourescent label and aphosphorylated amino acid, (ii) an antibody which selectively binds to amolecule comprising said phosphorylated amino acid; and (iii) asubstrate molecule comprising the same amino acid molecule that isphosphorylated in said reporter molecule; wherein said substratemolecule is labeled with an acceptor fluorophore and wherein saidantibody is labeled with a donor fluorophore which corresponds to theacceptor fluorophore labeling said substrate molecule.
 2. The article ofmanufacture of claim 1 wherein said phosphorylated amino acid isselected from the group consisting of serine, threonine and tyrosine. 3.The article of manufacture of claim 1 further comprising an enzymeselected from the group consisting of kinases and phosphatases.
 4. Thearticle of manufacture of claim 3 wherein the enzyme is a kinase.
 5. Thearticle of manufacture of claim 3 wherein the enzyme is a phosphatase.6. The article of manufacture of claim 1 further comprising a phosphatesource.
 7. The article of manufacture of claim 6 further comprising anenzyme selected from the group consisting of kinases and phosphatases.8. The article of manufacture of claim 7 wherein the enzyme is a kinase.9. The article of manufacture of claim 7 wherein the enzyme is aphosphatase.
 10. The article of manufacture of claim 1 furthercomprising a phosphate acceptor.
 11. The article of manufacture of claim10 further comprising an enzyme selected from the group consisting ofkinases and phosphatases.
 12. The article of manufacture of claim 11wherein the enzyme is a kinase.
 13. The article of manufacture of claim11 wherein the enzyme is a phosphatase.
 14. An article of manufacturecomprising, packaged together: (i) a reporter molecule comprising aflourescent label and a phosphorylated amino acid, (ii) an antibodywhich selectively binds to a molecule comprising said phosphorylatedamino acid; and (iii) a substrate molecule comprising the same aminoacid molecule that is phosphorylated in said reporter molecule; whereinsaid substrate molecule is labeled with an acceptor fluorophore andwherein said antibody is labeled with a acceptor fluorophore whichcorresponds to the donor fluorophore labeling said substrate.
 15. Thearticle of manufacture of claim 14 wherein said phosphorylated aminoacid is selected from the group consisting of serine, threonine andtyrosine.
 16. The article of manufacture of claim 14 further comprisingan enzyme selected from the group consisting of kinases andphosphatases.
 17. The article of manufacture of claim 16 wherein theenzyme is a kinase.
 18. The article of manufacture of claim 16 whereinthe enzyme is a phosphatase.
 19. The article of manufacture of claim 14further comprising a phosphate source.
 20. The article of manufacture ofclaim 19 further comprising an enzyme selected from the group consistingof kinases and phosphatases.
 21. The article of manufacture of claim 20wherein the enzyme is a kinase.
 22. The article of manufacture of claim20 wherein the enzyme is a phosphatase.
 23. The article of manufactureof claim 14 further comprising a phosphate acceptor.
 24. The article ofmanufacture of claim 23 further comprising an enzyme selected from thegroup consisting of kinases and phosphatases.
 25. The article ofmanufacture of claim 24 wherein the enzyme is a kinase.
 26. The articleof manufacture of claim 24 wherein the enzyme is a phosphatase.